Creatine kinase (also known as creatine phosphokinase, abbreviated CPK) is an enzyme that is present in the cells of heart muscle and other tissues of the human body, and it catalyzes the following reaction: ##STR1## where:
ADP=adenosine 5'-diphosphate
ATP=adenosine 5'-triphosphate
Measurement of the concentration of CPK in the circulating blood of patients suspected of having suffered a "heart attack" has proven to be of great value in the diagnosis of myocaridal infarction. A few hours after the occurrence of a myocardial infarction, CPK is released from the heart tissue into the circulating blood. Thereafter, the high circulating concentration of CPK decreases spontaneously over a period of several days returning to the low normal values. There are very few circumstances other than a myocardial infarction that result in elevated levels of CPK in the circulating blood. The detection of high levels of CPK therefore is indicative of the recent occurrence of a myocardial infarction.
The measurement of CPK is performed on a sample of the patient's blood that has been treated to obtain the blood serum or plasma. Because the quantity of enzyme in the serum or plasma is very minute even at elevated levels, the amount of enzyme is measured by its catalytic activity, i.e., its effect on the rate of reaction in vitro. Under certain conditions, the rate of reaction is directly proportional to the amount of CPK in the sample, and it can be measured by determining the rate of disappearance or appearance of one of the reactants or products.
The following is an example of a method currently in use for the determination of CPK that is based on the rate of formation of ATP, a product of the reaction. ATP can be measured by means of the following reactions [Kornberg, A., J. Biol. Chem. 182, 779 (1950)] ##STR2## where:
NAD=nicotinamide-adenine dinucleotide
NADH=nicotinamide-adenine dinucleotide reduced.
In the presence of appropriate concentrations of glucose, hexokinase, NAD, glucose-6-phosphate dehydrogenase and Mg++ in an aqueous solution, the rate of NADH formation is directly proportional to the rate of ATP formation, and thus to the quantity of CPK as described above. NADH absorbs light with an absorption maximum at 340 nanometers (nm) and this property can be used to measure the rate of formation of NADH. A procedure has been described, which measures CPK by determining the rate of increase of absorbance at 340 nm in an aqueous solution [Oliver, I. T. Biochem. J. 61, 116 (1955)]. This procedure is known as the "UV kinetic" method of analysis where UV stands for ultraviolet absorption associated with NADH.
The UV kinetic method offers the advantage that all ingredients necessary for the measurement of CPK are present in the reaction mixture but, at the same time, it suffers from several disadvantages. The application of this procedure to the rapid assay of large numbers of samples requires expensive, specialized equipment to measure the rate of change of absorbance. The sensitivity of the technique is limited by the relatively low molar absorbancy of NADH (Specifications and Criteria for Biochemical Compounds, Publication 1344, National Academy of Science, Washington, D.C. 1967). Because of the minute amounts of CPK present, the changes in absorbance are frequently so small as to tax the accuracy of the instrument used in making the measurement, thereby creating uncertainty in the values obtained for CPK.
Alternatively, one can carry out the enzyme catalyzed reaction for a definite period of time, stop the reaction and add a chromogenic reagent which reacts with one of the products to form a colored substance. This approach, known as the colorimetric method, has the following advantages: (1) total absorbance rather than rate of change of absorbance is measured, so that simpler measuring equipment can be used, (2) the enzymic reaction can be carried out for a longer period of time to enhance the amount of product formed, and (3) the chromogenic reagent can be selected to give a greater absorbancy than does NADH. The second and third conditions both offer the possibility of increasing the sensitivity of the test. Another advantage is that the measurement of the end product and the enzymic reaction need not be carried out concurrently, which makes it convenient to perform tests on a large number of samples.
The colorimetric method has been applied to the determination of CPK by other workers, but each of the methods described suffer from certain defects. Thus, in one of the methods currently in use, ADP produced in reaction I (run from right to left) is measured by means of the following reaction: ##STR3##
V. Pyruvate+2,4-dinitrophenylhydrazine.fwdarw.hydrazone [Nuttall, F. Q. and Wedin, D. S., J. Lab. Clin. Med. 68, 324 (1966)] This procedure suffers from the disadvantage that 2,4-dinitrophenylhydrazine does not react exclusively with pyruvate, and other compounds present in the sample or formed during the enzymic reaction can interfere.
Kuby, Noda, and Lardy, [J. Biol. Chem. 209, 191 (1954)], have described a procedure in which the creatine phosphate formed in reaction I (run from right to left) is measured after decomposing it to creatine and phosphate. The phosphate can be quantitated by conversion to a phosphomolybdic acid complex followed by reduction [Fiske, C. H. and Subbaow, Y., J. Biol. Chem. 66, 375 (1925)]. This procedure suffers from interference due to phosphate that is normally present in serum. Decomposition of ATP and ADP can also produce interfering phosphate.
The determination of either ATP or creatine in reaction I (run from left to right) has the advantage that the reaction from left to right reportedly occurs 2 to 5 times faster than the reverse reaction [Ennor, A. H. and Rosenberg, H., Biochemical J. 57, 203 (1954); J. A. Demetriou, P. A. Drewes, and J. B. Gin, Clinical Chemistry Principles and Technics (Second Edition), R. J. Henry, D. C. Cannon and J. W. Winkelman, Eds., Harper and Row, New York, N.Y., 1974, p. 898.], so that these methods are inherently more sensitive by that factor. Creatine may be measured by its reaction with diacetyl in alkaline solution in the presence of a phenol to enhance the color. Ennor and Rosenberg have described such a procedure in which the phenol was alpha naphthol. This method is subject to potential interference by substances that may be present in blood and the requirement for strong alkali is hazardous to the analyst.
In U.S. Pat. No. 3,929,580 a diagnostic reagent for the detection of CPK is prepared consisting of three individual compositions arranged in layers on a carrier and separated by physical barriers in order to impart stability to the reagent. Each of the layers must be adjusted to a limited range of pH values and pH range for one layer is not compatible with that in the other two layers.